Abstract Human coronaviruses are a diverse group of four antigenically distinct viruses that are important human pathogens that cause upper and lower respiratory tract infections. In young children and the elderly, lower respiratory tract infections can be severe. The recently emerged human coronavirus, SARS-CoV, causes severe pneumonia and death. It is a robust empirical model for identifying pharmacological probes of biological function that can strengthen our understanding of the proteins and pathways involved in establishment of disease. In addition, SARS-CoV is an ideal choice for the identification of broad spectrum antivirals for the treatment of coronavirus induced life threatening disease. In this proposal, we develop and validate novel assays, with plans for high throughput screening efforts during year 2 by the Molecular Libraries Production Centers Network (MLPCN). A yeast based assay will be used for the identification of compounds with activity against the SARS-CoV targets Nsp1 and PLP. Both targets give pronounced phenotypes when expressed in yeast, forming the basis for chemical screens to suppress the phenotypes. Important secondary screens will reveal hits that have anti-SARS-CoV activity, as well as those with broad spectrum activity against human coronaviruses in general. A pilot validation screen will be performed manually with the 2000 member Diversity Set library of compounds. The coronavirus model is robust because molecular clones exist, target genes have been identified that are essential for virus replication, structural information exists for both targets, and robust cell culture and murine models of severe end stage respiratory disease allow for biological evaluation of hits both in vitro and in vivo. In aim 1, we will optimize the expression and growth-inhibitory phenotypes of the Nsp1 and PLP proteins in yeast, characterize their behavior in 96-well plates, and perform the pilot screen. In aim 2, we will evaluate the antiviral activity of hits from the screen against recombinant SARS-CoV and other human coronaviruses encoding reporter genes. The ultimate goal of this proposal is to develop a rational, high throughput yeast-based screen that identifies small molecule inhibitors that target novel viral genes. The impact is high because our studies will develop a new high throughput paradigm to identify novel pharmaceuticals that will be used as molecular probes of coronavirus function. Equally important is that candidate broad spectrum antivirals against the target genes encoded within any microbial pathogen will also be identified. Project Narrative Experienced investigators at the University of Virginia and the University of North Carolina at Chapel Hill propose to develop new ways to discover chemical compounds ("drugs") that can be used to study the biology of the Severe Acute Respiratory Syndrome (SARS) coronavirus, called SARS-CoV. The budding yeast Saccharomyces cerevisiae will be used as a living test tube to measure the effects of specific SARS-CoV proteins on growth. Then, individual chemical compounds will be used to determine which compounds can reverse these effects. The idea here is that compounds that can reverse the effects of SARS-CoV proteins in yeast will also be able to reverse their effects in the human body during a SARS infection. We hypothesize that we can learn about the biology of SARS-CoV by identifying which chemical compounds can inhibit it, and then study how they work.